CS270195B1 - Murine Lymphocyte Hybridoma VU 809/5 - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing a monoclonal antibody against the gB glycoprotein of the Herpes simplex virus type 1, stored in the hybridoma collection of the Institute of Virology of the Slovak Academy of Sciences in Bratislava under the designation VÚ 809/5. The monoclonal antibody produced by the hybridoma VÚ 809/5 is suitable for diagnostic purposes in the immunofluorescence test /IF/ in the radioimmunoassay test /RIA/ and in the immunoenzymatic analysis /ELISA/ for determining the presence and amount of viral antigen in the tested material.

Description

The present invention relates to a novel hybridoma, i.e. a hybrid unicellular organism constructed by fusion of a mouse myeloma cell Sp2/0 and a mouse allesin lymphoid cell, producing an antibody to the gB glycoprotein of the Herpes simplex virus type 1.

In the Herpes simplex virus, we distinguish two antigenically different types of Herpes simplex type 1 and type 2. Until now, antibodies /antisera/ to the Herpes simplex type 1 virus have been prepared by immunizing experimental animals, most often rabbits, with purified or non-purified virus or some isolated protein'/Forghani B., Schmidt N., Lennette E.H.: Solid phase radioimmunoassay for identification of herpesvirus hominis types 1 and 2 from clinical material.'Appl. Microbiol. 2JB, /1974/ 661-667« Dreesman G.R., Watson 0.0., Courtney R.J., Adam E., Melnick J.L.i Detection of herpesvirus type-specific antibody by microsolid-phase radioimmunometric assayi Intervirology 12, /1979/ 115-119). The serum of animals thus immunized, collected after several doses of antibodies, is avidly anti-1AtoK so-called type-specific antibodies, which were used to detect the Herpes simplex virus type 1 antigen in basic research and in immunodiagnostic practice. Given that Herpes simplex viruses type 1 and. 2 contain not only type-specific but also type-common antigenic determinants, antibodies prepared in this way against the Herpes simplex virus contain! high levels of type-specific antibodies, which reduce or completely prevent! correct determination of the type of infecting virus in diagnostic testing. Type-specific antibodies are usually removed from the sera by saturation with Herpes simplex virus type 1, which is a procedure that is very demanding on the material and, moreover, successful only in a small number of cases. Production batches of conventional type-specific antisera are difficult to standardize and vary in quality. Recently, monoclonal antibodies, so-called type-specific antibodies, which are capable of detecting infection with Herpes simplex virus type 1 in clinical material, have been successfully used for typing Herpes simplex virus type 1.

The above disadvantages of the methods used so far will not occur if a hybridoma cell line producing a type-specific monoclonal antibody against the gB glycoprotein of the Herpes simplex virus type 1 is available, which is stored in the hybridoma collection of the Institute of Virology of the Slovak Academy of Sciences. Mlýnská dolina 1, Bratislava under the designation VÚ B09/5.

The above hybridoma was obtained by a method known from the professional literature (Kohler, G., Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, /1975/, 495., Gerhard, W. Fusion at cells in suspension and outgrowth of hybrids in conditioned medium. Monoclonal antibodies! A. new dimension in Biological analyses, Kennett R. H. et al., ads. New York, Plenum Press /1980/, 370.) Hybrid cells obtained after fusion of mouse myeloma Sp2/0 cells and cells obtained from the spleen of an OALD/c mouse immunized with an extract of cells infected with the Herpes simplex virus type 1 were cloned and after testing, clone VÚ 809/5 was selected. The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called. monoclonal antibody, which is capable of specifically reacting with the gB glycoprotein of the Herpes simplex virus type 1. Hybridoma VÚ 809/5 can be cultured in vitro in media suitable for animal cells or in vivo in the peritoneal cavity of BALB/o mice. From preserved frozen cells stored in liquid nitrogen, antibody production can be initiated without further immunization of the animal with antigen. '

Example

In order to obtain a larger amount of monoclonal antibody VÚ 809/5 by culturing hybridoma cells in vivo, 5x10 cells were applied to the peritoneal cavity of mice. For better cell attachment, the mice were premedicated with paraffin oil 15 days before the application of cells /0.5 ml intraperitoneally per 1 mouse/. After 10 days of hybridoma growth in the peritoneal cavity, the mice were killed and the produced ascitic fluid was collected. This procedure can obtain an average of about 7 ml of ascitic fluid containing B mg/ml of antibody. Ascitic fluid containing the product of hybridoma VÚ 809/5 showed specific binding to Herpes simplex virus type 1 in radioimmunoanalytical test /RIA/, in immunoenzymatic analysis /ELISA/ and in immunofluorescence test /IF/. By the method of radioimmunoprecipitation and electrophoresis in polar

CS 270 195 In acrylamide gel, the binding of the monoclonal antibody to glycoprotein gB of the Herpes simplex virus type 1 was detected.

Hybridoma cells VÚ 809/5 grow in vitro as a semi-suspension culture. They have a round shape and size characteristic of myeloma cells. They contain fused cell nuclei and are aneuploid. Hybridoma cells VÚ 809/5 have an ultrastructural picture of typical myeloma cells, where the predominant organelle is free and membrane-bound polyribosomes. The basic culture medium is Dulbecco's modification of Eagle's minimal essential medium (Dulbecco, R., Freeman, G., Virology 8 /1959/, 396). This medium, referred to as DMEM, is supplemented with gentamicin and inactivated precolostral calf serum (ΙΟΥ, Bioveta, Ivanovice ne Heně) for hybridoma cultivation. The hybridoma is cultivated at 37 °C in an atmosphere of 5¾ COj. Its generation time is approximately 24 hours. The antibody produced is a monoclonal immunoglobulin of the IgG 2a subclass.

Hybridoma VÚ 809/5 can be used as an eco-source of antibodies to the gB glycoprotein of the Herpes simplex virus type 1, which can be used as qualitative evidence of the presence of the Herpes simplex virus type 1 in the examined material, as quantitative determination of the amount of infecting virus or glycoprotein gB, when evaluating the epidemiological situation, for the purification of glycoprotein gB and as a source of antibodies of a single subclass (IgG 2a) for the preparation of antisera specific for the above subclass.

Claims (1)

Mouse lymphocyte hybridoma VÚ 809/5, producing a monoclonal antibody of the IgG 2a subclass against the gB glycoprotein of the Herpes simplex virus type 1.